The cross-talk at the prefronto-striatal interface involves excitatory amino acids, different receptors, transducers and modulators. We investigated long-term effects of a prepuberal, subchronic 5-HT7-R agonist (LP-211) on adult behaviour, amino acids and synaptic markers in a model for Attention-Deficit/Hyperactivity Disorder (ADHD). Naples High Excitability rats (NHE) and their Random Bred controls (NRB) were daily treated with LP-211 in the 5th and 6th postnatal week. One month after treatment, these rats were tested for indices of activity, non selective (NSA), selective spatial attention (SSA) and emotionality. The quantity of L-Glutamate (L-Glu), L-Aspartate (L-Asp) and L-Leucine (L-Leu), dopamine transporter (DAT), NMDAR1 subunit and CAMKIIα, were assessed in prefrontal cortex (PFC), dorsal (DS) and ventral striatum (VS), for their role in synaptic transmission, neural plasticity and information processing. Prepuberal LP-211 (at lower dose) reduced horizontal activity and (at higher dose) increased SSA, only for NHE but not in NRB rats. Prepuberal LP-211 increased, in NHE rats, L-Glu in the PFC and L-Asp in the VS (at 0.250 mg/kg dose), whereas (at 0.125 mg/kg dose) it decreased L-Glu and L-Asp in the DS. The L-Glu was decreased, at 0.125 mg/kg, only in the VS of NRB rats. The DAT levels were decreased with the 0.125 mg/kg dose (in the PFC), and increased with the 0.250 mg/kg dose (in the VS), significantly for NHE rats. The basal NMDAR1 level was higher in the PFC of NHE than NRB rats; LP-211 treatment (at 0.125 mg/kg dose) decreased NMDAR1 in the VS of NRB rats. This study represents a starting point about the impact of developmental 5-HT7-R activation on neuro-physiology of attentive processes, executive functions and their neural substrates. The interface between the prefrontal cortex (PFC) and the striata represents the neural substrate for the parallel processing of cognitive and non-cognitive information In the mammalian brain, the communication between the PFC and the dorsal/ventral striatum involves the amino acid L-Glutamate (L-Glu) acting through different ionotropic Therefore, in this manuscript we attempt to investigate long-term effects of a prepuberal, subchronic treatment, by LP-211, on adult behaviour, amino acid transmitters and synaptic markers, using a well documented rat model for ADHD By using a multidisciplinary approach, here we show that prepuberal LP-211 yields long lasting changes on adult behaviour, improving spatial attention in a dose dependent manner, associated with modified expression of pre- and post-synaptic markers.The results of the present experiments may represent an important starting point to explore how selective 5-HT7 receptor stimulation can impact upon the neurophysiology of attentive processes, executive functions and their neural substrates. This may, in turn, lead to a better understanding of developmental neuropsychiatric disorders that share activity, attention and executive function problems.To evaluate possible LP-211 developmental-treatment influences on body growth, the body weight was measured in all groups analyzed. There were no differences across groups in NHE and NRB rats. In fact, separate two-way factorial ANOVAs on NHE and NRB rats, with treatment x time (as covariate), showed no main effect for treatment but only for age  with no interaction.p<0.033) and for time  with no treatment per time interaction). The post-hoc LSD test confirmed that the reduction of HA produced by LP-211 was due to the 0.125 mg/kg dose (p<0.0064). On the other hand, the vertical activity in both NHE and NRB rats, VA and RD, were not influenced by prepuberal LP-211 treatment (one-way ANOVAs: no significant treatment effect on VA and RD) as was observed in the Làt maze  so that long-term effects of prepuberal subchronic LP-211 treatment could be determined. The behavioural tests consisted of short exposure of the rats to spatial novelties paradigms, i.e. Làt and Olton mazes. The main variables were total activity, non selective and selective spatial attention , and emotionality index in Broadhurst's terms Làt maze .p<0.024). Post-hoc LSD test showed that the higher score was due to the 0.250 mg/kg dose of LP-211 (p<0.0087). In contrast, the other parameter, i.e. the total Number of alley Visits needed To Criterion (NVTC), was not different among groups, as demonstrated by one-way ANOVA. The defecation score, used as an emotionality index in Broadhurst terms, was not different across treatment groups and behavioural tasks (Làt and Olton mazes), as demonstrated by separate Kruskal–Wallis tests within the NHE and NRB rats.For the selective spatial attention (SSA), the two markers of non-reinforced SSA are depicted in ex-vivo levels of the amino acids L-Glu, L-Asp and L-Leucine in the PFC, DS and VS by LC/MS/MS. The choice of L-Asp was based on its hypothesised role in neurotransmission as suggested by D'Aniello et al. It is very well known the role of excitatory amino acids in the synaptic dialogue between the pre-frontal cortex (PFC) and the dorsal/ventral striatum (DS/VS) Excitatory amino acids L-Glu and L-Asp were expressed as a ratio over L-Leu that does not participate into neurotransmission. Moreover, they were measured in the soluble fraction that corresponds to the one sampled by the microdyalisis method Separate one-way ANOVAs on NHE and NRB rats showed an increased ratio of L-Glu/L-Leu  for LP-211 at 0.250 mg/kg dose in the NHE line . ConversIn the DS, separate one-way ANOVAs on NHE and NRB rats revealed a decreased L-Glu/L-Leu ratio only for the 0.125 mg/kg dose  in the NHE line. In contrast, no significant difference was observed in the levels of L-Glu/L-Leu in the NRB line. Furthermore, the L-Asp/L-Leu ratio was also reduced at the 0.125 mg/kg dose  in the NHE rats. No significant changes were observed across amino acids and LP-211 doses in the NRB rats .Finally, the levels of L-Glu/L-Leu in the VS of NHE rats showed no significant differences. Separate one-way ANOVAs on NHE and NRB rats demonstrated a reduced L-Glu/L-Leu ratio in the VS for the 0.125 mg/kg dose , only in NRB rats . MoreoveLong term changes in neural substrates were detected using, as molecular probes, the synaptic transmission markers dopamine transporter (DAT), NMDAR1 subunit and calcium-calmodulin-dependent kinase type II alfa (CaMKIIα). DAT has been chosen as presynaptic marker In the PFC, the one-way ANOVA on the DAT level of NHE rats, treated with vehicle, 0.125 mg/kg or 0.250 mg/kg LP-211, showed a significant reduction at 0.125 mg/kg dose  . This reThe goal of these experiments was to verify whether the higher density of the NMDAR1 subunit, found in the PFC of NHE rats, could be modified by stress-associated vehicle injections and/or LP-211 treatment at adolescence. NMDAR1 protein level in the PFC of NHE and NRB rats, under basal conditions, are shown in NMDAR1 protein levels in the brain areas  of NHE rats, treated with vehicle, 0.125 mg/kg or 0.250 mg/kg LP-211, are shown in The spatio-temporal coincidence detector CaMKIIα The rationale for these studies was to investigate whether and to what extent a prepuberal subchronic stimulation of serotonin 7 subtype receptors (5-HT7-R), by the selective LP-211 agonist, would produce long term effect on adult behaviour, amino acid transmitters and synaptic markers. This was carried out using a validated animal model of hyperactivity and attention deficit, the NHE line, and compared to the NRB control rats.A prepuberal subchronic design was chosen based on the assumption that i) the prepuberal period is “critical” for the organisation of neural networks in the developing forebrain, and that these sites are sensitive to genotype x environment interactions By using a multi-disciplinary approach, we show that adolescent exposure to LP-211 yields long lasting changes on adult behaviour, improving spatial attention in a dose dependent manner, associated with modified pre/post synaptic markers.Prepuberal subchronic treatment apparently modified neural circuits underlying locomotor activity, NSA and SSA, as monitored in adulthood by exposure to two spatial novelty paradigms (i.e. Làt and Olton mazes). Under basal conditions, the NHE rats showed a higher frequency of leanings than NRB rats In particular, the stimulation of 5-HT(7) receptors could alternatively have been induced by a mixed 5-HT(1A/7) agonist with the help of a 5-HT(1A) selective antagonist ex vivo tissues, the compartmentalisation of excitatory amino acids in the mammalian brain. The soluble form includes amino acids that can be washed out with cold saline together with part of the neuro-glial component. Amino acids are distributed in three main heterogeneous pools, for which a homeostasis has been proposed by Kalivas The ratio of Glutamate and Aspartate over Leucine, an amino acid not involved in neuro-transmission, pertained only to the soluble forms and was comparable to that measured in micro-dialysis The presynaptic DAT, the post-synaptic NMDAR1 and CaMKIIα markers, studied under basal conditions, showed that NMDAR1 levels within the PFC of NHE rats were higher, in comparison with NRB rats; by contrast, no differences were found within the DS. Moreover, NHE rats showed a significant reduction in the amount of CaMKIIα within the PFC, while previous findings Treatment with LP-211 during adolescence gave the following findings that persisted into adulthood: 1) DAT level in the PFC was decreased at the 0.125 mg/kg dose and conversely increased in the VS at the 0.250 mg/kg, but only in NHE rats. The adolescent prepuberal treatment produced no differences in all areas of NRB control subjects; 2) NMDAR1 was decreased in the VS of NRB rats when previously faced with the prepuberal subchronic exposure to a 0.125 mg/kg dose; 3) prepuberal subchronic LP-211 exposure exerted no long-term effect on CaMKIIα levels in either line and in all areas. It is noteworthy that CaMKIIα level has been reported to be lower in the forebrain of animal models for ADHD, such as SHR and NHE rats Altogether, the most relevant change was produced by the 0.250 mg/kg dose of LP-211: adolescent exposure to this dose generated, as a sequel into adulthood, an improved spatial orientation as well as persistently elevated L-Asp and DAT levels in the VS and L-Glu levels in the PFC. The simplest interpretation of these results is that the cross-talk between the PFC and VS might have been improved.The “dialogue” between the PFC and both the DS and VS, occurring through glutamate and aspartate, is modulated mainly by DA, NA, 5-HT and histamine. Our experimental evidence suggests that only two signaling modulators (DA and 5-HT) are effective in inducing long-term changes within neural networks, which are known to underlie attentive and motivational processes. In fact, prepuberal subchronic treatments with MPH and ATX, which block the reuptake of DA/NE and NE, respectively, did not similarly alter the SSA in adult rats, in spite of significant effects on DA, NA, 5-HT and metabolites at the prefronto-striatal interface in NHE rats This profile of effects is likely to be explained because DA has the following functions: i) at mesencephalic level, DA can interact with DA-D2 autoreceptors, associated with hyper-polarising potassium conductances, thereby reducing the firing rate of DA neurons The fact that the developmental stimulation of 5-HT7 receptors (by the selective agonist LP-211) is just as effective as DA-related drugs in modifying the long-term SSA in NHE rats requires further experimental investigations. One clue is given by the fact that 5-HT7 is found to be functionally linked to forebrain limbic networks, which comprise the amygdala, VS, orbital PFC, and thalami Therefore, we can assume that the positive effect of adolescent exposure to 0.250 mg/kg LP-211 on SSA, in association with enhanced levels of amino acids and DAT in the PFC and VS, is possibly due to the stimulation of serotonergic more effective transducers independent of CaMKIIα. Alternatively, this positive effect might be explained by more complex circuits, leading - eventually - to the re-modeling of cognitive maps: in this context, recent findings Finally, the concentration of CaMKIIα Interestingly, recent findings from our laboratory do point to a disintegrated interface in the PFC of NHE rats, as monitored by immuno-chemical markers 2; Techniplast, Italy) under standard conditions. An artificial 12∶12 reverse light–dark (LD) cycle was obtained by turning on the lights from 7 pm to 7 am. All other parameters fulfilled the requirements of the Guide for the Care and Use of Laboratory Animals, implemented by the Italian Legislative Decree 116/1992 that covers all scientific procedures involving the use of live animals. The European Directive 2010/63 on the protection of animals used in scientific experiments, revisioning the Directive 86/609, is currently being enacted into Italian legislation, and will be applicable to future research protocols. All present experiments were covered by an authorisation from Ministero della Salute, for which ethical advice was issued from Istituto Superiore di Sanità . The 3Rs principles were taken into account in order to seek for alternatives to animal use, to reduce the number of animals used and to refine the protocols for limitation of animal suffering.Prepuberal male rats of the Sprague–Dawley-derived NHE line (n = 24) and of the NRB control line (n = 24), from our animal colony, were used throughout the experiments. Rats were housed in groups of two inside Makrolon cages  28, rats were randomly assigned to treatment and control groups (n = 6/group), which received i.p. vehicle or LP-211 . LP-211 was dissolved in phosphate buffer (vehicle) with 1% and 2% DMSO for the lower doses and the highest one, respectively. Treatments were given daily during the dark phase between 10:00 am and 11:00 am, for 14 days from pnd 30–31 to 43–44, as already published A new selective 5-HT7 receptor agonist, namely N-(4-cyanophenylmethyl)-4-(2-diphenyl)-1-piperazinehexanamide (LP-211), was synthesized and provided by EL and ML R), closed by a cover of the same material. A smaller transparent-plastic 30 cm×30 cm×40 cm box was inserted in the middle, thus providing a 60 cm long, 15 cm wide and 40 cm high square-shaped exploratory corridor. See 2. A set of two such boxes was located in a sound attenuated experimental room.The Làt-maze consisted of a 60 cm×60 cm×40 cm box made of special poly-vinyl-chloride (PVC) material (KÖMACELAt the beginning of the dark phase of an inverted light/dark L/D cycle (between 9 am and 4 pm), the rats were individually exposed to the Làt-maze and allowed to explore the corridor for 10 min. In order to minimise the interference with the arousal state, pairs of rats from the same home cage were tested at the same time. The animals' behaviours were monitored by a high resolution CCD camera and stored on a DVD recorder to be analyzed off-line. The number of corner crossings (HA), frequency (VA) and duration (RD) of rearings on hind-limbs, or leanings against the walls with one or both forepaws, were measured in 1-min blocks. The reliability index was quite high . At the end of the test, the fecal boluses were counted and the floor was wiped with a wet sponge.The apparatus consisted of eight arms (8 cm×60 cm) extending from an octagonal centre platform (18.5 cm diameter). The distance from the platform centre to the end of each arm was 69.25 cm. The apparatus was constructed of grey PVC with a smooth surface, and side walls . The maze was placed on the floor of a dimly lit room, surrounded by a circular higher wall without visual cues. Rat behaviour was monitored by a high resolution CCD camera and stored on DVD to be analysed off-line.In the Olton's maze, each rat was placed on the centre platform into a cardboard cylinder to avoid immediate escape into an arm. The trial began with the removal of the cylinder and the rat was allowed to explore. The main parameters considered in the Olton's maze were horizontal activity ; frequency (VA) and duration (RD) of rearings on the hind-limbs. The number of arms visited before the first repetition occurred , and number of arms visited before completion of visits to all eight arms , were also recorded. The latter two indices are markers of non-reinforced SSA.The Rat Brain Atlas as reference The animals were sacrificed by a guillotine at pnd 77. The brain was removed and put on a surface of ice-cold saline. After removal of the olfactory tubercles, the first coronal cut was made at 4.20 AP from Bregma, using the stereotaxic coordinates of Vehicle, LP-211 0.125 and 0.250 mg/kg groups were analysed. The 0.500 mg/kg group was excluded because it did not show any significant effect in the behavioural analysis. Brain samples were homogenised in 1 ml ice-cold saline in an Eppendorf and centrifuged at 7,500 g at 4°C for 20 min. The supernatant was filtered and used for LC/MS/MS analysis of the free amino acids L-Glu, L-Asp and L-Leu.18column  at a flow rate of 0.3 mL/min. The mobile phase consisted of aqueous 0.1% formic acid (A) and acetonitrile (B). Samples were eluted with a linear gradient from 10 to 90% B in 5 min. At 5:01 min, solvent B was decreased from 90% to 10% and remained constant for 5 min. The total run time was 10 min.The detection was performed using a Varian 310-MS triple quadrupole mass spectrometer . All the analytes were detected in the positive ionisation mode and with selected reaction monitoring (SRM) mode Table 1. Membrane and cytosolic protein fractions were prepared as described in Sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) and transfer of proteins to nitrocellulose membranes were performed according to conventional methods with minor modifications. The proteins were solubilised in loading buffer at 54°C for 45 min, and then separated on 10% polyacrylamide gels and transferred onto PVDF membranes  in transfer buffer . The membranes were incubated with blocking buffer, 5%ECL blocking agent  in PBST , for 1 h at room temperature. The blots were incubated overnight at 4°C with one of the following primary antibodies: NMDAR1 ; CaMKIIα ; DAT ; internal standard: Actin ; or Actin .After three 10 min washes in PBST, the blots were incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody . After washing, immuno-complexes were visualised by chemiluminescence using the ECL western blotting kit  according to the manufacturer's instructions. All protein bands were within linear range of the standard curves, and were normalised to actin levels on the same membrane. Quantity One software  was used for standardisation and quantisation of protein bands obtained by western blot analysis.The fulfilment of the requirements for parametric analysis  was assessed by specific tests  and appropriate transformations were used when necessary. Behavioural variables in the Làt and Olton's radial maze were submitted to a one-way factorial analysis of variance (ANOVA) for treatment followed by post-hoc test LSD for single comparison.p>0.05 after correction of the alpha-level by the Hölm's procedure Defecation scores were submitted to a non-parametric test by Kruskal–Wallis. The rejection level was set at


This demonstrates that endogenously derived NO can modulate both the timing and intensity of locomotor-related activity. The effects of DEA/NO were mimicked by the cGMP analog 8-bromo-cGMP. In addition, the soluble guanylyl cyclase (sGC) inhibitor ODQ blocked the effects of DEA/NO on burst amplitude and frequency, although the frequency effect was only blocked at low concentrations of DEA/NO. This suggests that NO-mediated modulation involves cGMP-dependent pathways. Sources of NO were studied within the lumbar spinal cord during postnatal development  with NADPH-diaphorase staining. NOS-positive cells in the ventral horn exhibited a rostrocaudal gradient, with more cells in rostral segments. The number of NOS-positive cells was also found to increase during postnatal development. In summary, we have shown that NO, derived from sources within the mammalian spinal cord, modulates the output of spinal motor networks and is therefore likely to contribute to the fine-tuning of locomotor behavior.Spinal motor control networks are regulated by neuromodulatory systems to allow adaptability of movements. The present study aimed to elucidate the role of nitric oxide (NO) in the modulation of mammalian spinal locomotor networks. This was investigated with isolated spinal cord preparations from neonatal mice in which rhythmic locomotor-related activity was induced pharmacologically. Bath application of the NO donor diethylamine NONOate (DEA/NO) decreased the frequency and modulated the amplitude of locomotor-related activity recorded from ventral roots. Removal of endogenous NO with coapplication of a NO scavenger (PTIO) and a nitric oxide synthase (NOS) blocker [nitro- The ability to produce rhythmic activity needed for locomotion is an inherent property of the spinal cord. The spinal motor network that produces this rhythm is termed the locomotor central pattern generator (CPG). The locomotor CPG is in turn regulated by a range of neuromodulatory systems that enable locomotor output to be adaptable . Three isoforms of NOS have been identified , with the function of two of these variants, NOS1 and NOS3, being activity dependent because of their requirement for calcium/calmodulin binding. NO typically acts via the stimulation of soluble guanylyl cyclase (sGC) to increase intracellular levels of cGMP, which can act upon a range of intracellular and membrane-bound proteins . Isolated tissues were pinned down and the dorsal and ventral roots trimmed. Prepared tissues were then transferred to a recording chamber superfused with aCSF with a flow rate of 5–8 ml/min. For histological analyses spinal cord tissue was prepared from P1–P12 C57BL/6 or CD1 mice, as described above for physiological preparations, and tissues were then fixed via submersion in 4% paraformaldehyde (wt/vol) in 0.1 M phosphate buffer  for 12 h. The aCSF used for both dissections and recordings contained (in mM) 127 NaCl, 26 NaHCO3, 10 glucose, 3 KCl, 2 CaCl, 1.25 NaH2PO4, and 1 MgCl2.The use of animals in this investigation was in accordance with the United Kingdom Animals (in Scientific Procedures) Act 1986 and was reviewed and approved by the University of St Andrews Animal Welfare and Ethics Committee. For physiological preparations spinal cord tissue was prepared from postnatal day (P)2–P5 C57BL/6 mice, as described previously . In brie1/L2), to record flexor-related activity, on both left and right sides at identical segmental levels of isolated spinal cord preparations (obtained from P2–P5 C57BL/6 mice). In a subset of experiments, extensor-related activity was also recorded from the fifth lumbar ventral root (L5). Rhythmic locomotor-related activity, alternating between contralateral upper lumbar roots and ipsilateral L2 and L5 roots, was induced pharmacologically with a combination of N-methyl-d-aspartic acid  and 5-hydroxytryptamine , with or without dopamine (50 μM). The inclusion of dopamine was associated with lower burst frequencies in control . However, statistical analyses demonstrated that the effects of subsequent drug applications did not differ between preparations with and without dopamine. For experiments investigating the importance of excitatory versus inhibitory components of the network we utilized a disinhibited preparation (n = 16), picrotoxin: 6.2 ± 1.0 a.b. (n = 14), Student's t-test, P > 0.05; root-mean-squared (RMS) amplitude: bicuculline 0.48 ± 0.07 a.b. (n = 16), picrotoxin: 0.50 ± 0.08 a.b. (n = 14), Student's t-test, P > 0.05; frequency: bicuculline 0.061 ± 0.004 Hz (n = 16), picrotoxin: 0.054 ± 0.004 Hz (n = 14), Student's t-test, P > 0.05]. Subsequent drug applications were made once the bursts of locomotor-related activity recorded from ventral roots had stabilized . Rectified/integrated traces were used for the detection of locomotor-related ventral root bursts. The instantaneous frequencies, peak-to-peak amplitudes, and RMS amplitudes (for disinhibited preparations) of ventral root bursts were then measured from raw traces. Amplitude was measured as a noncalibrated unit and is therefore presented as an arbitrary unit (a.b.). For time course plots data were aggregated into 1-min bins, or 2-min bins for disinhibited preparations, and normalized to a 10-min precontrol period to facilitate comparison between preparations. To calculate burst duration, individual bursts from a 10-min smoothed trace (4 iterations of 200 ms moving average) were downsampled to 100 Hz and the duration of each burst (the time taken from 40% peak amplitude on the upstroke to 40% peak amplitude on the downstroke) was calculated with a custom “R” script. Duty cycle was calculated as burst duration divided by cycle period. Statistical comparisons of burst amplitude and frequency were performed on absolute means, calculated over 5-min periods for standard preparations and 6-min periods for disinhibited preparations, for control, drug application, and washout phases. Data were analyzed with repeated-measures ANOVA and pairwise comparison with a Tukey's honestly significant difference (HSD) test, with 2) ventral root bursts (at their peak amplitude) while the individual data points indicate the mean phase of ipsilateral extensor-related (L5) root bursts . Center of mass, defined as the time point at which the voltage reached half of its cumulative summed total, was chosen for the L5 bursts because of the elongated shape of their extensor-related activity. Arrows associated with the circular plots indicate the mean direction of the phase relationships across all preparations analyzed.The phase relationship between activity recorded from different pairs of ventral roots was assessed with circular plots and Rayleigh's test for uniformity . CompariAfter fixation, lumbar segments (1st to 5th) were cryoprotected in 30% sucrose (wt/vol) in PB at 4°C for 12 h and then frozen and stored at −20°C until being sectioned. Tissues were cut into 10- to 15-μm transverse sections on a cryostat and mounted on glass slides. Slides were incubated at 37°C with 1 mg/ml β-NADPH, 0.13 mg/ml nitro blue tetrazolium (NBT), and 0.3% Triton X-100 (vol/vol) in 0.1 M phosphate-buffered saline  for 110 min and then washed in PBS to halt the reaction. In the presence of NOS and NADPH, NBT is reduced to the alcohol-insoluble blue dye nitro blue tetrazolium formazan, thereby indirectly labeling NOS-expressing cells for visualization under light microscopy .n = 3) and CD1 (n = 24) mouse strains were used for histological analyses, no differences in the distribution of NADPH-diaphorase-positive cells were observed between the two strains. Given that the density of spinal neurons reduces developmentally, we adjusted cell counts to account for changes in lumbar spinal cord volume with age. The volume of the lumbar spinal cord was calculated from measurements of fixed spinal cord tissue . Lumbar volume estimates were calculated as the volume of a cylinder using the mean spinal cord diameter at L1, L3, and L5 and the mean length between L1 and L5 . With “R” and the package “MASS,” count data were fitted with a negative binomial generalized linear model using age and lumbar position as factors and log volume as an offset term (thereby fitting the model to counts per mm3 of volume). The influence of the factors age and lumbar position were assessed with likelihood ratio tests.Animals were categorized by age into four equally spaced groups . With reference to Rexed's laminae, the dorsal horn was defined as lamina I–VI, the ventral horn as lamina VII–IX, and the area proximal to the central canal as lamina X. For each animal, images of five nonconsecutive (separated by at least 30 μm) spinal cord slices were obtained for each lumbar segment of interest. The number of positively stained cells in the dorsal horn, lamina X, and ventral horn were then counted manually. Although both C57BL/6  in PBS with 1% bovine serum albumin (wt/vol) and 0.1% Triton X-100 (vol/vol). Bound primary antibody was labeled with fluorescein isothiocyanate (FITC)-tagged donkey anti-rabbit secondary antibody  during a 2-h incubation. Z-plane optical sections of the stained tissue were created with an epifluorescence microscope and structured illumination , and the images were processed into a maximum Z projection .Two days prior to experimentation CD1 mice received an intraperitoneal injection of Fluoro-Gold  to retrogradely label sympathetic preganglionic neurons (SPNs) . Spinal l-arginine methyl ester (l-NAME), the sGC inhibitor 1H-oxadiazoloquinoxalin-1-one (ODQ), and 5-HT were obtained from Abcam . All other reagents were of analytical grade and supplied by either Fisher Scientific UK  or Sigma-Aldrich. All drugs were dissolved in reverse osmosis water, except 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO) and ODQ, which were dissolved in DMSO, and stored in aliquots at −20°C. Final concentration of DMSO in the working solutions was <0.1% (vol/vol).The NO donor diethylamine NONOate (DEA/NO) was supplied by Cambridge Bioscience . The cGMP-dependent protein kinase activator 8-bromo-cGMP was obtained from Tocris Bioscience . The NOS inhibitor nitro-n = 15 preparations; 100 μM, n = 13; 200 μM, n = 17; 400 μM, n = 10) to isolated spinal cord preparations while recording pharmacologically induced fictive locomotor activity from ventral roots . The modulatory effects of DEA/NO on the frequency of the locomotor rhythm were concentration dependent , with a maximum reduction observed at a dose of 400 μM. Given the variability in control frequencies of different preparations, we investigated whether there was a relationship between the starting frequency and the magnitude of the effect induced by DEA/NO; however, no significant relationship was identified.To investigate whether NO-mediated signaling can modulate the activity of spinal motor networks controlling mammalian locomotion, we bath applied the NO donor DEA/NO at a range of concentrations . Duty cycle was found to decrease with 200 μM and 400 μM DEA/NO , but no significant change was observed with lower concentrations .In a subset of experiments we also assessed the effects of DEA/NO on burst duration and duty cycle. Burst duration was unchanged at all concentrations of DEA/NO . In contrast to the consistent reduction in burst frequency, the polarity of the effect of DEA/NO on burst amplitude was concentration dependent. At higher concentrations (200 and 400 μM) DEA/NO caused a reduction in burst amplitude . However, at lower concentrations the application of DEA/NO either had no effect  or led to an increase in burst amplitude . While the reduction in burst amplitude induced by high concentrations of DEA/NO was reversible, the increase in burst amplitude induced by 50 μM DEA/NO remained after drug washout and persisted for the duration of stable recordings (up to 60 min after initiation of washout). Control experiments in which no drugs were applied (beyond those used to elicit locomotor activity) demonstrated that locomotor burst amplitude is typically stable over a similar time course .DEA/NO also induced a concentration-dependent change in the amplitude of locomotor-related ventral root bursts  alternation remained unaltered . This was demonstrated by circular phase plots in which the mean direction of left-right  and flexor-extensor  alternation was unchanged by the application of DEA/NO (50–200 μM DEA/NO). The mean resultant length of the vectors for both left-right and flexor-extensor relationships was found to decrease in the presence of DEA/NO , demonstrating an increase in the variability of the phase relationships. This reduction in the strength of phase relationships is likely to result from an increase in the variability of locomotor output as revealed by greater coefficients of variation for frequency during the application of DEA/NO at all concentrations tested  = 4.2, P < 0.05; control/drug/wash: F = 2.6, P > 0.05; interaction: F = 5.9, P < 0.001, n = 7–10].While the frequency, amplitude, and duty cycle of locomotor-related activity were modulated by DEA/NO, mean phase relationships of left-right  and flexor-extensor  while recording fictive locomotion from isolated spinal cord preparations. When l-NAME and PTIO were coapplied to reduce the amount of endogenous NO available, we observed a decrease in the amplitude of locomotor-related ventral root bursts . Interestingly, the washout of l-NAME and PTIO was associated with an increase in amplitude during an extended washout period . The similarity between this increase and the long-term effects of 50 μM DEA/NO application suggest that it may relate to a rebound in endogenous NO levels following the washout of blockers/scavengers. The coapplication of l-NAME and PTIO also led to an increase in the frequency  of locomotor-related activity. There was again no relationship between control frequency and the magnitude of frequency effects induced by l-NAME and PTIO. These findings demonstrate that NO is produced by the spinal cord during locomotor network activity. Furthermore, this endogenous NO modulates locomotor control circuitry to regulate the frequency and intensity of locomotor-related output.Next we investigated whether NO-mediated modulation represents an endogenous mechanism utilized by spinal cord circuitry to control locomotor output. To investigate the potential role of endogenous NO in the modulation of the murine locomotor network, we coapplied the NO scavenger PTIO (400 μM) and the NOS inhibitor Cii; F = 16.3, P < 0.001, n = 9) and a sustained increase in the amplitude of locomotor-related ventral root bursts . The actions of 8-bromo-cGMP mimic the effects of the lower concentration of DEA/NO  and support involvement of the sGC/cGMP pathway in the modulatory actions of NO on the murine locomotor control network.NO-mediated signaling typically involves the activation of NO-sensitive sGC and the subsequent accumulation of intracellular cGMP that can act on a range of intracellular and membrane-bound targets . To elucF = 1.8, P > 0.05, n = 7, data not shown; DEA/NO 200 μM: F = 2.3, P > 0.05, n = 12, Ci). ODQ also blocked the reduction in frequency induced by 50 μM  but not 200 μM  DEA/NO. Comparison of the percent change in frequency induced by DEA/NO (200 μM) with and without ODQ (100 μM) showed no difference in the magnitude of this effect . At higher concentrations of ODQ  the locomotor rhythm became unstable or ceased, so we could not test the possibility that 100 μM ODQ was an insufficient concentration to block the effects of 200 μM DEA/NO on locomotor frequency. Together these findings further support the idea that NO-mediated modulation of the intensity of locomotor output generated by spinal CPG circuitry involves the sGC/cGMP pathway.To further elucidate the role of the sGC/cGMP pathway we next applied DEA/NO (50 or 200 μM) in the presence of the sGC blocker ODQ (100 μM). The effects of both 50 and 200 μM DEA/NO on the amplitude of locomotor-related activity were blocked by ODQ  or the GABAA channel blocker picrotoxin (60 μM). Ventral root recordings from these preparations revealed slow, rhythmic, large-amplitude bursts that were synchronous across segmentally aligned left and right ventral roots . However, l-NAME and PTIO had no effect on the peak-to-peak amplitude of locomotor-related bursts in disinhibited preparations . Given the irregular shape of disinhibited bursts, with a sharp peak followed by a lower-amplitude plateau, we also utilized RMS amplitude as a measure of the intensity of motor output. However, there was again no change in RMS amplitude with the application of l-NAME and PTIO . These findings indicate that endogenous NO modulates the frequency of locomotor activity via effects on excitatory components of the locomotor control network, while modulation of burst amplitude likely involves inhibitory components.To begin to explore the spinal components involved in NO-mediated modulation of locomotor control circuitry, we utilized disinhibited preparations  to pharmal roots A. In keeCii; F = 8.4, P < 0.01, n = 11), and although there appeared to be a trend toward an increase in burst amplitude, this did not reach statistical significance whether measured as peak-to-peak or RMS amplitude . In agreement with application of 50 μM DEA/NO, 200 and 400 μM DEA/NO also produced a decrease in the frequency of locomotor-related activity . However, in contrast to 50 μM DEA/NO, 200 and 400 μM DEA/NO produced a decrease in RMS burst amplitude  but no change in peak-to-peak amplitude .We next assessed the effects of exogenous NO on disinhibited preparations. The application of 50 μM DEA/NO to disinhibited preparations caused a decrease in the frequency of locomotor-related activity  and the control frequency , with slower initial burst frequencies associated with smaller DEA/NO-induced decreases in frequency. This may suggest there is a minimum frequency at which the already slow disinhibited preparations can generate rhythmic output.For disinhibited preparations we again assessed whether the magnitude of the frequency effects induced by PTIO and These findings suggest that NO modulates the frequency of locomotor-related activity by affecting excitatory components of CPG circuitry. In contrast, the cellular components involved in NO-mediated modulation of the intensity of locomotor-related output appear to vary depending on the source and concentration of NO.n = 6), P4–P6 (n = 6), P7–9 (n = 6), and P10–P12 (n = 9). At each of these age groups the number of NADPH-diaphorase-positive cells was assessed within the ventral horn, lamina X , and the dorsal horn (1–L5). All cell counts were adjusted to the estimated lumbar volume for each age group.Having established that endogenous NO derived from spinal sources modulates the activity of spinal locomotor circuitry, we next investigated the potential sources of this NO and their postnatal development. The distribution of NO sources within the lumbar spinal cord was elucidated by using NADPH-diaphorase staining to indirectly label cells expressing NOS over the postnatal developmental period P1–P12 , A and Bsal horn  from theNADPH-diaphorase-labeled neurons with intensely stained somata and proximal dendrites were found across all age groups and all lumbar segments , A and B2 = 36.4, P < 0.001). Analyses across different age groups also revealed an increase in NADPH-diaphorase-positive neurons in the ventral horn with age .Perhaps most relevant to locomotor control, NADPH-diaphorase-positive neurons were consistently observed within the ventral horn of the lumbar spinal cord. Labeled cells were scattered throughout lamina VII and VIII but were rarely seen within motoneuron pools (lamina IX), with less than one cell per animal in lamina IX for each lumbar segment D. Cell c present . The ros2 = 23.8, P < 0.001). However, in contrast to the ventral horn, the number of NADPH-diaphorase-positive cells in lamina X did not change with age . Thus the gradient observed in lamina X along the entire lumbar cord reflects the presence of SPNs in upper lumbar segments. To demonstrate the presence of NOS-positive interneurons among SPNs in lamina X of the upper lumbar cord, we also performed double-fluorescence labeling experiments using Fluoro-Gold to retrogradely label SPNs and NOS1 immunohistochemistry to label nitrergic neurons. Consistent with previous reports  but were found to increase with the age of animals  burst durations along with decreases in duty cycle may relate to whole animal studies showing that during changes in locomotor speed the flexor (swing) phase remains relatively constant while the extensor (stance) phase is altered . Furthermore, several studies have demonstrated that modulation of locomotor-related frequency does not necessarily coincide with changes in amplitude, and vice versa  or 8-bromo-cGMP to the isolated spinal cord led to a sustained increase in the amplitude of locomotor-related activity. These long-lasting effects, which extended into the washout period and remained as long as stable rhythms could be recorded, may represent an LTP-type effect of NO on the locomotor control network. Similar effects were also seen during an extended washout of pocampus  and, morpocampus . In addipocampus . Our finIn addition to examining the physiological effects of NO-mediated signaling on spinal motor circuitry, we also investigated spinal sources of NO and their postnatal development by using the NADPH-diaphorase labeling technique. The pattern of NOS-positive neurons demonstrated in this investigation are in agreement with those shown previously in the adult rat with NADPH-diaphorase histochemistry  and NOS In agreement with developmental findings in the rat lumbar spinal cord , we founIn agreement with previous studies , we findIn summary, we have demonstrated that endogenous NO, derived from sources within the developing mouse spinal cord, modulates locomotor-related output produced by spinal motor circuitry. At concentrations we speculate are most physiologically relevant, NO-mediated modulation includes long-term changes to the intensity of locomotor-related output. Meanwhile, experiments investigating the effects of higher, possibly pathological, concentrations of NO highlight potentially deleterious inhibitory effects this modulator may have on motor output. While advancing our understanding of the neuromodulatory control of spinal motor networks, this study also provides a platform from which to investigate the influences of NO at the cellular level. Such work is not only important for further advancement of our basic understanding of motor control but may also highlight pathways that can be targeted for the treatment of injury and disease affecting the spinal cord.Wellcome Trust (Grant no. 091177/Z/10/Z). C. Dunford was supported by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship.This work was supported by the No conflicts of interest, financial or otherwise, are declared by the author(s).Author contributions: J.D.F., C.D., and G.B.M. conception and design of research; J.D.F. and C.D. performed experiments; J.D.F. and C.D. analyzed data; J.D.F., C.D., K.T.S., and G.B.M. interpreted results of experiments; J.D.F. and C.D. prepared figures; J.D.F. and G.B.M. drafted manuscript; J.D.F., C.D., K.T.S., and G.B.M. approved final version of manuscript; C.D., K.T.S., and G.B.M. edited and revised manuscript.


An analysis of all known human herpesviruses has not previously been reported on sperm from normal donors. Using an array-based detection method, we determined the cross-sectional frequency of human herpesviruses in semen from 198 Danish sperm donors. Fifty-five of the donors had at least one ejaculate that was positive for one or more human herpesvirus. Of these 27.3% (n = 15) had a double herpesvirus infection. If corrected for the presence of multiple ejaculates from some donors, the adjusted frequency of herpesviruses in semen was 27.2% with HSV-1 in 0.4%; HSV-2 in 0.1%; EBV in 6.3%; HCMV in 2.7%; HHV-6A/B in 13.5%; HHV-7 in 4.2%, whereas none of the samples had detectable VZV or HHV-8. Subsequently, we examined longitudinally data on ejaculates from 11 herpesvirus-positive donors. Serial analyses revealed that a donor who tested positive for herpesvirus at one time point did not necessarily remain positive over time. For the most frequently found herpesvirus, HHV-6A/B, we examined its association with sperm. For HHV-6A/B PCR-positive semen samples, HHV-6A/B could be detected on the sperm by flow cytometry. Conversely, PCR-negative semen samples were negative by flow cytometry. HHV-6B was shown to associate with sperm within minutes in a concentration dependent manner. Confocal microscopy demonstrated that HHV-6B associated with the sperm head, but only to sperm with an intact acrosome. Taken together, our data suggest that HHV-6A/B could be transported to the uterus via binding to the sperm acrosome. Moreover, we find a 10 times higher frequency of HHV-7 in semen from healthy individuals than previously detected. Further research is required to determine the potential risk of using herpesvirus-positive donor semen. Longitudinally analyses of ejaculate series indicate that implementation of quarantine for a donor shown to shed a herpesvirus is not a tenable solution. Semen is a known vehicle of infectious agents. Human herpesviruses have previously been detected in human semen, albeit with varying frequencies in different studies. Most studies have used sperm from men attending fertility clinics, but whether herpesviruses impair fertility is controversial and thus it remains an open question whether data from this population reflects the frequency of herpesviruses in semen from the general population. A recent study suggested that although human cytomegalovirus (HCMV) may produce a gametotoxic effect, the frequency of infectious activity was not different among semen from infertile and fertile men The initial analysis on the presence of all herpesviruses on German men seeking fertility evaluation by Bezold et al. Notably, studies from Greece on either normal semen samples or semen samples from men attending fertility clinics report much higher frequencies, in particular of EBV (16.8–45.0%) and HCMV (7.1–62.5%) Although their presence has been established, the localization of most of the herpesviruses in semen has yet to be determined. _ENREF_16 It has been reported that HSV-2 and HCMV can associate with the sperm membrane Since herpesviruses are characterized by their ability to cause latent infections that may later recur, we wished to determine the frequencies of herpesviruses in Danish donor semen, in addition to their presence in semen during longitudinal analyses.The study was conducted according to the Helsinki declaration and sperm donors had signed a declaration of consent stating that the donated semen may be used for scientific intentions. Samples were anonymized and data are not referable to the donor. The Ethical Committee of Science for the Region of Middle Jutland has approved the study (M-20100238).http://dk.cryosinternational.com/clinics/screening.aspx), previous known genital infections, current infection of C. trachomatis or N. gonorrhoae (swab), or sero-positivity towards human immunodeficiency virus type1 and 2, human T-cell lymphotropic virus type 1 and 2, hepatitis B and C, and T. pallidum. The ethnicity of the donors reflects that of the general Danish population of young men Semen samples were obtained from Cryos International Sperm Bank in Aarhus, Denmark. The age of the donors at the time of sperm donation ranged from 18 to 48 years, with a mean age of 26 years. Donors had been examined and excluded if they had a predisposition to certain inheritable diseases  and the robotic QiaCube (Qiagen) according to the manufacturer’s instructions.Herpesviruses were detected by a Clart®Entherpex kit  according to the manufacturer’s instruction. This kit identifies all known human herpesviruses, but does not discriminate between HHV-6A and HHV-6B. When the specific type of HHV-6 is not known, we will refer to it by HHV-6A/B. The Entherpex kit is based on amplification of a type-specific DNA fragment using primers that recognize highly conserved regions. Two different sets of biotinylated primers were used to amplify the specific fragments, which were hybridized to type-specific probes on the array. Amplified complimentary viral DNA fragments were visualized with the use of a streptavidin-peroxidase conjugate and o-Dianisidine. The platform includes an internal amplification control, which provides information of potential PCR-inhibition. Results were obtained using an Array CCD camera-reading device , with automated interpretation and alignment software.6 per ml. HHV-6B was prepared as previously described 2b , followed by a secondary chromophore-conjugated goat anti-mouse (GaM) antibody . The samples were then washed twice, dissolved in PBS/1% paraformaldehyde, and 50,000 sperm per sample were analyzed on a Beckman-Coulter FC500 Analyzer. From the samples prepared for flow cytometry analysis, an aliquot of 10 µl was analyzed on a Zeiss confocal microscopy with a 63× magnification. For the analysis of HHV-6B binding to sperm acrosomes, sperm were treated with dimethyl sulfoxide (DMSO) diluted 1∶100 or 10 µM calcium ionophore  at 37°C for 30 min, then washed and incubated at 37°C with 400 IU/ml HHV-6B for 1 hr. Samples were washed, stained and incubated with 1% paraformaldehyde as described above, only this time with co-staining using rhodamine-conjugated pisum sativum (PSA) .HHV-6B binding to sperm was assessed by flow cytometry and confocal microscopy. Sperm samples were washed, resuspended in PBS, and purified with PureSperm  when indicated. Washed or purified sperm were resuspended in PBS to a concentration of 8 x 10Ejaculates (n = 318) from a total number of 198 different donors were analyzed for the presence of human herpesviruses. Fifty-five of the donors had at least one ejaculate that was positive for one or more human herpesvirus. Of these 27.3% (n = 15) had a double herpesvirus infection. Since more than one ejaculate was examined for 56 of the donors, these donors had an increased probability of a positive sample. We therefore calculated the frequency by adjusting for the number of ejaculates. Thus, a donor was weighed by the fraction of positive ejaculates, ie. the number of positive ejaculates divided by the total number of ejaculates from that donor. Calculated this way, the adjusted frequency for herpesvirus in donor sperm was 27.2%.Similar adjustments for the individual human herpesviruses resulted in the following frequencies: HSV-1 in 0.4%; HSV-2 in 0.1%; EBV in 6.3%; HCMV in 2.7%; HHV-6A/B in 13.5%; HHV-7 in 4.2%, whereas none of the samples had detectable VZV or HHV-8 .In order to determine whether an ejaculate from a donor with a previously positive ejaculate can rightfully be assumed positive, series of ejaculates collected over a time period of 4 years were analyzed longitudinally from 11 herpesvirus-positive donors. This demonstrated that donors shed herpesviruses intermittently, in accordance with what is known for recurrent herpesvirus infections , and thuThe most prevalent herpesvirus in semen was HHV-6A/B, of which HHV-6B is the variant known to be shed in secretions in vivo.Notably, even in vehicle-treated semen samples, a low intensity binding of gp60/110 was detectable. This was not caused by unspecific antibody binding to sperm, since a single straw of sperm that tested negative for HHV-6B by PCR showed only anti-gp60/110 antibody binding if HHV-6B was added in vitro . We therThe detection of HHV-6B association to sperm by flow cytometry, prompted us to examine whether the virus bound to specific regions of the sperm. Sperm were incubated with HHV-6B and prepared for confocal microscopy . SpecifiThe potential serious consequences of congenital infections highlights the importance of decreasing transmission of infectious agents to the mother, the fetus or the newborn. Sperm is a possible transmission route, in particular for the mother. Herpesviruses are one family of viruses with several members of importance for sexually transmitted infections. HCMV is well known for its ability to cause serious congenital infections. Although not complied with by the Danish sperm banks, guidelines for gamete donations presented by the American Society for reproductive Medicine (ASRM) in 1998 included screening of donors for HCMV IgG and IgM Additional research is needed to address the issue of whether screening of donor ejaculates for human herpesviruses should be required of sperm banks before the ejaculate is released. Our analysis of ejaculate series strongly implies that implementation of quarantine for a donor shown to shed a virus is not a tenable solution. Donors with a sperm sample positive for a herpesvirus may later become non-detectable for the same virus, which again may recur in a later sample. This suggests that each sperm sample rather than the donor should be characterized for the presence of virus.We find that HHV-6A/B is the most prevalent herpesvirus among donors from Denmark. HHV-6A/B is a beta-herpesvirus and thus related to HCMV. Whether HHV-6A/B may cause some of the same problems for pregnant women as is known for HCMV is poorly understood. We found that HHV-6B is able to bind to the acrosome of the sperm, but it is still an unsolved question how herpesviruses come into contact with the mature sperm. This might occur in the testis during spermatogenesis or from an infection of the urogenital tract. Although Naumenko et al. The consequences of herpesviruses on sperm quality or human reproduction, if any, are still debatable. However as long as we are not certain that these viruses are harmless, we must acknowledge the risk of viral transmission through semen. In particular, whether HHV-6B may be transmitted to the oocyte by means of the sperm requires further analysis. Our finding that HHV-6B binds to the acrosome may argue against this possibility, since the acrosome is dissolved when the sperm interacts with the egg. Nevertheless, HHV-6A/B has been reported to be chromosomally integrated We used a sensitive PCR test to determine the frequency. However, our longitudinal analysis indicated that donors might change their status of herpesvirus secretion with time. Since virtually all adults are HHV-6A/B seropositive, it is possible that almost everybody may secrete HHV-6A/B in semen at some point, although the level may vary considerably between individuals, which may be important. Clearly, it becomes a challenge to determine the potential consequences, if any, of the intermittent shedding of a common virus.Importantly, the binding of HHV-6B appeared to be specific since it was only seen on sperm with acrosomes. Despite using a high titer of HHV-6B, HHV-6A/B was detected by flow cytometry on sperm that were also HHV-6A/B positive by PCR. Moreover, approximately one out of seven men secretes at a given point HHV-6A/B in semen. Thus, it is not surprising that the vehicle-treated sample in Besides HHV-6A/B, our study finds the related virus HHV-7 in approximately 1 out of 24 semen samples from healthy donors. To our knowledge, HHV-7 has not previously been detected in semen from normal donors and thus its significance, if any, is unknown. Bezold et al. found that 0.4% of German men seeking fertility evaluation had HHV-7 in their semen In summary, our study has identified several herpesviruses, of which HHV-6A/B was the most predominant, in semen from healthy donors. The virus appears to be transmitted bound to the acrosome of sperm. Moreover, we demonstrate that the presence of herpesviruses may fluctuate, even for the same donor, probably reflecting recurrence of the latent infection.


Estrogens play an important role in modulating the morphology and function of temporomandibular joints (TMJs), which is suggested to act via estrogen receptors (ERs). The present study was to investigate the expression of aggrecan, collagen type II (Col II), Col X, aromatase, ERα and ERβ in degenerative changes of mandibular condylar cartilage.Forty male and 40 female 8-week-old rats were enrolled in this study. In experimental groups, the disordered occlusion was created by moving the first molars mesially and the third ones distally. Immunohistochemistry and real-time PCR were performed at the end of the second or fourth week.Degenerative changes, characterized by interrupted continuity of hypertrophic layer, pyknotic and eosinophilic lesion with few nuclei, areas filled with eosinophilic nuclei, were observed in more joints from female experimental groups than male ones. However, thickening changes in hypertrophic layer were only found in male experimental groups. The gene expression of Col II, Col X and aggrecan increased in 4-wk male experimental subgroup (both P < 0.01), but decreased in 2-wk and 4-wk female subgroups (P < 0.05). The gene expression of ERα decreased in 2-wk male and female experimental subgroups (both P < 0.01), however, that of ERβ increased except the 2-wk female experimental subgroup . The expression of aromatase decreased in both male and female experimental subgroups .Mandibular condylar cartilage responses differently to the disordered occlusion in male and female rats. The levels of locally synthesized estrogen, ERα and ERβ may have limited attribution, if any, to the sex-specific cartilage response. Temporomandibular disorders (TMD) is a collective term that embraces a number of clinical conditions that involve the masticatory musculature, temporomandibular joint (TMJ) and associated structures. Many epIn literature, various studies have shown that estrogens synthesized by aromatase-expressing bone cells may have important effects at the skeletal tissue level, independent of the circulating levels of estrogens,17. Our TMJ is a load-bearing structure with its load mainly from elevators, the function of which is adjusted by occlusion through feedback from mechanoreceptors in periodontal tissues. The articular surface of the TMJ is covered by fibro-cartilage that is distinct from the hyaline cartilages of other joints. It is aThe study was approved by the Animal Research Committee of the Fourth Military Medical University. Forty male and 40 female 8-week-old SD rats, weighing 200-220g in male and 180-200g in female, were provided by the animal center of the Fourth Military Medical University. All animals were randomly divided into experimental group and control group, twenty for each group.All animals were housed under conditions of 22°C and 30-60% relative humidity with a normal day-night rhythm, consisting of a 12:12 hour light–dark cycle. The rats had free access to tap water and standard food, and no dietary adjustments were made. Before the beginning of the experiment, all the animals were bred for 2 days to adapt to the environment.TM Elastics, 3M Unitek, 1/8#), about 1 mm in diameter, was inserted between the first and second molars of the left side of maxillary dentition and the right side of mandibular dentition. The rubber bands were carefully placed at the level lower than the occlusal surface of the molars so that occlusal contact relationship would not be interfered. The first molars were gradually moved mesially, with a relatively stable gap about 0.8 mm wide 7 days later. Then, to keep this gap till the end of the experiment, the elastic bands were replaced by self-curing resin . Care was taken to prevent the resin to contact with opposing molars. At the beginning of the 5th week, the same method was used to move the left maxillary and right mandibular third molars distally. In all the occlusal treatments, the length of mouth opening was less than 15mm, with the time of each operation less than 5 minutes. In control group, all the rats were subjected to all the procedures, except for inserting the rubber bands or placing self-curing resin between the molars. Animals in experimental group and their age-matched controls were sacrificed at the end of the second or fourth week after the inserting the last rubber band, named as the 2-wk and 4-wk subgroups. During our observed experimental period, there was no significant difference in feed intake or body weight between experimental and sham groups.In experimental group, occlusal treatment, which has been proved to induce obvious degenerative changes of mandibular condylar cartilage successfully, was carried out with a method as previously reported,30. BrieUnder deep anesthesia with intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight), for morphological analysis and immunohistochemistry, four rats in each group were perfused with 200ml normal saline through ascending aorta, followed by 400 ml paraformaldehyde . The TMJs were dissected and post-fixed overnight at 4°C with the same fixative, and then decalcified for 1 week in Kristensen’s fluid . TMJ samples were then dehydrated in graded ethanol and then embedded in paraffin. Serial mid-sagittal sections of 5 μm in thickness were cut parallel to the lateral surface of the condyle neck of the mandible ramus. For histological observation, one section from each joint was stained with hematoxylin and eosin. For the real-time PCR analysis, the other six rats in each group were sacrificed. To get enough total RNA, four condylar cartilages from 2 randomly selected rats were considered as one sample, so 12 condylar cartilages from 6 rats in each group were randomly assigned into 3 samples.All measurements were made by one examiner (Lei Liu) who knew nothing about the grouping of animals. The sections stained with HE were observed and photographed under microscope (Leica DM 2500). A true-colour computer-assisted image analyzing system with a digital camera  and software  were applied for images capture and measurement. As we previously reported, the totFive commercially available primary antibodies were used in immunohistochemical staining. They were anti-human Col II goat polyclonal IgG , anti-rat Col X rabbit polyclonal IgG , anti-human ERα rabbit polyclonal IgG , anti-rat ERβ rabbit polyclonal IgG , and anti-human aromatase goat polyclonal IgG . Four sections from the middle part of each joint were selected for this study. Immunohistochemical staining was carried out with a three-step avidin-biotin complex method as described previously. BrieflyTM First Strand cDNA Synthesis Kit (Fermentas). Real-time PCR was performed in ABI 7500 Fast thermal cycler. The protocol comprised 40 cycles of 94°C for 5 sec, 62°C for 34 sec, and 72°C for 1 min each. The detected cytokines were Col II, Col X, aggrecan, ERα, ERβ and aromatase. Table-▽▽CT and reported as arbitrary units[Mandibular condylar cartilage samples were pulverized in liquid nitrogen. The total RNA was isolated from frozen tissues using a standard TRIzol® protocol , followed by first-strand cDNA synthesis with the RevertAidry units.The results were quantified or evaluated by one examiner (Lei Liu) who knew nothing about the grouping of animals. The SPSS 11.0 package  was used to analyze and describe the data. Three-factor analysis of variance with full interaction was adopted. The fixed factors were treatment (control and experiment), time point (2-wk and 4-wk), and sex . When significant main effects or the interaction between main effects were found, specific comparisons between groups were made by Student’s t test. For the results of the ranked data about the percentages of OA-like degenerative regions, were compared by using the nonparametric Mann–Whitney U test. P-values were considered to be statistically significant when less than 0.05.In mandibular condylar cartilage of control group, fibrous layer, proliferating layer, mature layer and hypertrophic layer arranged regularly with good continuity in each layer Figurea,1b. HoIn mandibular condylar cartilage of control group, the Col II and Col X immunohistochemical signals distributed continuingly in mature and hypertrophic layers, especially in hypertrophic layer Figurei and1j.Compared to their age-matched control subgroups, the gene expression of Col II, Col X and aggrecan increased significantly in 4-wk male experimental subgroups , but decreased significantly in both 2-wk and 4-wk female experimental subgroups (P<0.05). The gene expression of Col II, Col X and aggrecan in 4-wk male experimental subgroups was higher than 2-wk male experimental subgroups (P<0.05), while that in 4-wk female experimental subgroups was lower than 2-wk female experimental subgroups (P<0.05). Additionally, the gene expression of Col II and Col X in 2-wk male experimental subgroups, and that of Col II, Col X and aggrecan in 4-wk male ones, was significantly higher than female age-matched experimental subgroups  , but that of ERβ increased significantly in 2-wk male and 4-wk both male and female experimental subgroups (both P<0.01). However, the expression level of ERβ in 2-wk female experimental subgroup was significantly lower than its age-matched control group. Additionally, the gene expression of ERα and ERβ in 4-wk both male and female experimental subgroups was higher than 2-wk sex-matched experimental subgroups (P<0.05). Furthermore, the gene expression of ERβ in 2-wk male experimental subgroups, was significantly higher than female age-matched experimental subgroups (P<0.01). No such sex differences were found between other subgroups . The authors declare that they have no competing interests.Meiqing Wang led the project and revised the manuscript. Shibin Yu carried out the histology, immunohistochemistry staining, and drafted the manuscript. Xianghui Xing carried out real-time PCR, and revised the manuscript. Kai Jiao performed the statistical analysis and helped to revise the the manuscript. Lei Sun carried out the animal model. Lei Liu carried out the animal model, and evaluated the results. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/13/190/prepub


La) on mammalian cell growth, recombinant protein production, and environmental conditions of the culture .Orbitally shaken flasks are commonly used at an early stage of bioprocess development with mammalian cells. In contrast to large-scale stirred-tank bioreactors, shaken flasks are usually operated in probe-independent bioprocesses, i.e. without strictly controlling the pH or dissolved oxygen concentration (DO). As a consequence, gas transfer issues are thought to limit the effectiveness of orbitally shaken flasks and bioreactors (OSRs). To define optimal operating conditions for probe-independent bioprocesses in OSRs, we tested the effects of the mass transfer coefficient of oxygen . A recombinant CHO DG44-derived cell line expressing a human IgG monoclonal antibody (CHO-IgG) [La on cell growth, CHO-IgG cells were into 1-L cylindrical bottles with working volumes from 200 to 600 mL. The bottles were equipped with vented caps and orbitally shaken at 110 rpm in an incubator at 37°C with 5% CO2. To test the kLa as a scale-up factor, CHO-IgG cells were inoculated at 0.3 million cells/mL in a 200-L OSR  with a working volume of 100 L and agitated at 57 rpm. Air containing 5% CO2 was flushed into the OSR at 1 L min-1. After overnight cultivation, samples were withdrawn from the 100-L culture and used to inoculate satellite cultures in 1- and 5-L bottles with vented caps. The volume of the cultures in bottles was adjusted to obtain the same kLa as the one in the 200-L bioreactor (7 h−1), and the bottles were agitated at 110 rpm.The kribed in  using noCHO-IgG)  was cultLa decreased from 11 to 3 h-1 as the working volume increased from 200 to 600 mL , the maximal cell density was about 40% less than in cultures of ≤ 400 mL . The recombinant IgG concentrations at this time were about 150 mg/L. The pH decreased from 7.25 to 6.7 in all the cultures (data not shown), and the glucose, glutamine, lactate and glutamate profiles were similar in all the cultures.To test the kLa is a good parameter to predict suitable conditions for cell cultures in probe-independent OSRs. Furthermore, our study demonstrates that cultures having different nominal scales but the same kLa also had the same cell growth, recombinant protein production, and culture conditions (pH and DO). The minimal kLa required to avoid pH and DO limitations in OSRs was 7 h-1 for CHO-IgG cells. Cell cultivation in a 200-L OSR without pH or DO controllers resulted in similar cell densities, recombinant protein titers and pH values as in 1- and 5-L OSRs when the three types of OSRs were operated at the same kLa. These results suggest that large-scale bioprocesses can be operated without pH or DO controllers as long as a sufficient kLa is maintained through appropriate cultivation conditions .Our results indicate that the k


We demonstrated that overexpression of miR-101 sensitized A549 cells to CDDP, one of the most frequently used agents in curing or controlling NSCLC and enhanced CDDP-induced cell death and caspase 3-dependent apoptosis. In addition, miR-101 facilitated the inhibitory role of CDDP in A549 cell colony formation. Overall, the results of the present study demonstrated that miR-101 sensitizes the A549 NSCLC cell line to CDDP via the activation of caspase 3-dependent apoptosis.MicroRNA-101 (miR-101) is evidently downregulated in several types of cancer, including non-small cell lung cancer (NSCLC), and is crucial in sensitizing cells to chemotherapy drugs. The aim of the present study was to investigate the correlation between miR-101 and chemosensitivity in the A549 NSCLC cell line. Here, we used the human A549 cell line for transfection with an miR-101 overexpressing vector and detected the cytotoxic acticity, proliferation and apoptosis of  The underlying mechanism has not been determined  cells by inhibition of mammalian target of rapamycin (mTOR) signaling via proline-rich Akt substrate 40 (PRAS40)  are a class of small, single-stranded, non-coding RNAs of 19–25 nt that are involved in the regulation of cellular development, proliferation, differentiation, apoptosis and metabolism . miRNAs er cells . Notablyer cells ,19. In a(PRAS40) . Xu et autophagy . HoweverIn the present study, we provide further evidence that miR-101 enhances the chemosensitivity of A549 NSCLC cells to CDDP. Furthermore, miR-101 was shown to significantly promote CDDP-induced apoptosis and suppress the colony formation by activating the caspase 3-dependent apoptosis pathway.2 at 37°C. Transfection of A549 cells with plasmids, pre-miR-101 or scrambled pre-miR control , was performed using Lipofectamine 2000 according to the manufacturer's instructions . Untransfected A549 cells were employed as a negative control.The A549 cell line was derived from adenocarcinomas of the lung obtained from the China Center for Type Culture Collection . The lung cancer cells (A549) were maintained in RPMI-1640 medium  with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 μg/ml streptomycin) within a humidified atmosphere containing 5% CO−ΔΔCt method.Total RNA was extracted from cells using a modified TRIzol one-step extraction method (Invitrogen Life Technologies). Stem-loop reverse transcription for mature miR-101 and U6 primers was performed as previously described. U6 RNA was used as an miRNA internal control. The primers used for stem-loop reverse-transcription PCR for miR-101 were purchased from Guangzhou RiboBio Co., Ltd. . Each sample was conducted in triplicate and the results were calculated using the 23 cells/well and allowed to grow overnight. Cells were then treated with various concentrations of CDDP . Following 24 h of CDDP treatment, 20 μl MTT  was added to the cells, which were incubated in the dark for 4 h. Following the removal of the cell supernatants, formazan crystals were dissolved in 150 μl dimethylsulfoxide. The viability of treated cells was calculated from the average OD570 values compared with those of the untreated cells with an enzyme-linked immunosorbent assay reader . Each group was run in triplicate wells.The effect of miR-101 on cell growth was measured by MTT assay. A549 cells that had been mock-transfected or transfected with pre-miR-101 were seeded respectively into 96-well plates at a density of 5×10A549 cells were transfected and treated with CDDP. Following 24 h of treatment, cells were reseeded into six-well plates with a density of 500 cells per well for 2–3 weeks. The medium was discarded and each well was carefully washed twice with phosphate-buffered saline (PBS). The colonies were fixed in methanol for 20 min and then stained with Giemsa staining solution. The number of colonies with ≥50 cells were counted and colony forming efficiency was calculated using the following formula: Percentage of colonies (%)= number of colonies formed/number of cells inoculated ×100. Experiments were repeated three times.6 cells/ml. In total, 100 μl cell suspension (1×105 cells) was transferred to a 5-ml culture tube. Following this, 10 μl PI was added to the cell suspension and incubated for 15 min at room temperature in the dark. The stained cells were then diluted to 500 μl with binding buffer and analyzed with a FACSCalibur flow cytometer . Triplicate assays were performed.Cell death was assessed by flow cytometry using propidium iodide (PI). A549 cells were transfected and treated with CDDP as described above. The cells from various treated groups were collected and washed twice with cold PBS. The cells were then resuspended in binding buffer at a concentration of 1×10To evaluate the degree of CDDP-induced apoptosis, TUNEL assays were performed. A549 cells were incubated in six-well plates and transfected as previously described. Following 24 h of transfection and then 24 h with or without CDDP stimuli exposure, the cells in each well were washed twice with cold PBS and fixed with 4% (v/v) formaldehyde in PBS for 10 min at room temperature. TUNEL was performed using the In Situ Cell Death Detection Kit, Fluorescein  according to the manufacturer's instructions. Randomly selected microscopic fields (n=8) were counted under a fluorescent microscope  and the ratio of TUNEL-positive cells to the total number of cells was calculated in three independent experiments using ImageJ software .A549 cells from various treated groups were washed twice in cold PBS and lysed for 20 min using RIPA buffer , with freshly added protease inhibitor and phosphatase inhibitor . Protein concentration in the cell lysate was determined using the BCA assay . In total, 50 μg of protein was separated on SDS-PAGE and then transferred onto PVDF membranes . Next, the membranes were blocked with 2% BSA in TBST containing 0.1% Tween-20 for 1 h at room temperature. Membranes were then incubated overnight at 4°C with antibodies against caspase 3,  and β-actin . After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody  followed by ECL detection .All statistical analyses were conducted using SPSS 11.5 software . All numerical data were generated from three independent experiments and are presented as means ± standard error of the mean. Differences between groups were examined using one-way analysis of variance or Student's t-test, as appropriate. P<0.05 was considered to indicate a statistically significant difference.In order to explore the role of miR-101 in A549 cells, transfection with plasmids, pre-miR-101 or scrambled pre-miR control, was performed. The expression levels of miR-101 were confirmed by qPCR, as shown in In order to explore the manner in which the modulation of miR-101 in the A549 cell line affected CDDP-induced cell death, pre-miR-101 transfected cells and the Con and miR-Con groups were treated with CDDP and analyzed by flow cytometry. The results of the flow cytometry revealed that the mean rates of cell death were 1.3, 39.6, 41.4 and 93.0% for the Con, CDDP, CDDP plus miR-Con and CDDP plus miR-101 groups, respectively . These rTo explore whether miR-101 expression may alter CDDP-induced apoptosis, apoptosis was measured by TUNEL staining. As shown in To confirm the long-term survival rate of cells, a colony assay was performed in the A549 cell line with various treatments as described previously. As shown in Although chemotherapeutic agents, including CDDP, are widely used in the treatment of lung cancer, their efficacy is often limited by the existence or development of chemoresistance. Factors that enhance the sensitivity of NSCLC cells to chemotherapeutic drugs may highlight predictive biomarkers or targets for therapy. miR-101 is an miRNA, which has been previously demonstrated to regulate a variety of biological processes by modulating the expression of several target genes ,23. A gret al reported that miR-101 enhances the chemosensitivity of PDAC cells by inhibition of mTOR signaling via PRAS40 (et al reported that miR-101 enhanced apoptosis induced by CDDP in the HepG2 cell line by inhibiting autophagy (To date, the mechanism by which miR-101 enhances chemosensitivity remains unclear. Previously, Batchu a PRAS40 . Xu et autophagy . In ordeIn conclusion, the present study demonstrated for the first time that miR-101 functions as an inducer of chemotherapeutic sensitivity in NSCLC by activating the caspase 3-dependent apoptosis pathway. These results established that miR-101 transfer in combination with CDDP therapy may be a target to reverse chemotherapeutic resistance. Further investigations with regard to the miR-101 regulation of chemosensitivity are likely to provide insights into the mechanistic details of this regulatory network.


Internal resistances times volume were decreased, even though internal resistances times membrane surface area were not. Since the membrane in the flat-plate design is placed vertically, membrane surface area per geometric planting area is increased, which allows for lower internal resistances times volume while not decreasing internal resistances times membrane surface area. Anode was split into three different sections on different depths of the system, allowing to calculate internal resistances on different depths. Most electricity was produced where internal resistances were lowest and where most roots were present; in the top section of the system. By measuring electricity production on different depths in the system, electricity production could be linked to root growth. This link offers opportunities for material-reduction in new designs. Concurrent reduction in material use and increase in power output brings the P-MFC a step closer to usable energy density and economic feasibility.Due to a growing world population and increasing welfare, energy demand worldwide is increasing. To meet the increasing energy demand in a sustainable way, new technologies are needed. The Plant-Microbial Fuel Cell (P-MFC) is a technology that could produce sustainable bio-electricity and help meeting the increasing energy demand. Power output of the P-MFC, however, needs to be increased to make it attractive as a renewable and sustainable energy source. To increase power output of the P-MFC internal resistances need to be reduced. With a flat-plate P-MFC design we tried to minimize internal resistances compared to the previously used tubular P-MFC design. With the flat-plate design current and power density per geometric planting area were increased (from 0.15 A/m Currenting area ,maximum  in 2008  to 220 m in 2010  in systeing area . In someing area . In one ing area .Power density of a P-MFC is determined by several aspects of the system: solar radiation, photosynthetic efficiency of the plant, organic matter allocation from plant to soil, and efficiency of the Microbial Fuel Cell (MFC) . Timmersmax = 1.1 V), current density  and internal resistance  in a P-MFC [Equation 1 Power density (P) as function of maximum voltage  on day 320 of the experiment Figure . In bothIn case of P-MFC 1 this was true; most roots were found in the top part of the system, less in the middle part and least in the bottom part. Anodic resistance of the bottom part of P-MFC 1 Figure a, b incrP-MFC 2 shows a different pattern Figure c, d. TopThe high anode resistance in our experiment is in contrast with earlier research with the tubular system, which showed a high membrane resistance, which was mainly caused by the transport resistance ,14. In o+ or Na+) to travel to the cathode is larger than in the flat-plate system  has a large influence on the internal resistance. In our experiment plant-roots were mainly found in the top part of the system, which was consistent with previous experiments ,11,12,14m Figure .3 it shows that Timmers et al. produced 7.5 A/m3 and 1.3 W/m3 whereas we produced 122 A/m3 and 5.8 W/m3. Firstly, this leads to the conclusion that materials are more efficiently used in the flat-plate system than in the tubular system, leading to higher outputs on volume base. Secondly, when normalizing current and power density to geographic planting area 1.6 A/m2 and 0.44 W/m2 were achieved, which is higher than reported for a tubular P-MFC  in a-1 in the experiment of Timmers et al. and 1.1-1.3 S.m-1 in our experiment. Higher conductivity in our experiment will lead to a lower ionic resistance compared to Timmers et al. This would mean, however, that according to Equation 5 transport resistance could be expected to be higher than in the case of Timmers et al. The use of buffer in the experiment of Timmers et al. and the lack of it in our experiment can have an influence on the pH-gradient over the membrane. It is shown in Figure In the tubular system a large transport resistance was found, whereas transport resistance in the flat-plate system is low. It should be noted though, that the plant-growth medium can influence transport resistance as well. Conductivity was 0.15-0.17 S.mTimmers et al. have shown that the time-steps used to make a polarization curve have a large influence on the internal resistance . The lonWhen comparing the obtained power and current densities of the flat-plate P-MFCs with the expected results based on Equation 1, it shows that only in a few cases measured value approximates theoretical value Table . This haThe flat-plate P-MFC design resulted in a lower transport and membrane resistance than the previously used tubular P-MFC. It did, however, not result in a lower total internal resistance normalized to membrane surface area. Total internal resistance of the flat-plate P-MFC is at best comparable to total internal resistance of the tubular P-MFC, only differently distributed over several partial internal resistances. In the flat-plate P-MFC the anodic resistance is highest, when using a chemical cathode, due to substrate limitation or mass transfer limitation. To overcome the problem of substrate limitation, either the plant should exudate more, exudates should be converted into electricity more efficiently or other rhizodeposits, like dead root material should be used. Possibly, when rhizosphere is fully mature, more dead root material is available, which will probably lead to higher substrate availability in the P-MFC. Higher exudation rates might be achieved by adapting the plant-growth medium to stimulate exudation . For futSpartina anglica ) were planted in each P-MFC. The anode compartment was separated from the cathode compartment by a cation exchange membrane (CEM) .Two flat-plate P-MFCs were run for 703 days. The anode compartment had a total volume of 648 ml (18x18x2 cm). The anode consisted of three sections of graphite felt of 5 cm height , which were physically separated Figure  A gold w3Fe(CN)6, 50 mM) was used as catholyte to stabilize cathode-potential and be able to study anode-potential. Ferric cyanide was replenished whenever cathode-potential dropped below +200 mV (against Ag/AgCl reference electrode).The cathode consisted of three sections of graphite felt of 5 cm height, , each corresponding with an anode section Figure . The catFrom day 1 through day 116 top, middle and bottom sections of the anode were electronically connected, thus behaving as one anode. During this period external load between anode and cathode was 300 Ω. From day 117 anode-sections were electronically disconnected. The three anode-sections all were connected to their corresponding cathode over an external load of 900 Ω.Different plant-growth media were used during the runtime of the experiment, as described in Helder et al. 2011 for P-MFC 3 and 4 . At deteAnode and cathode potential were measured with Ag/AgCl-reference electrodes . Data was logged every 60 seconds with Fieldpoint  and collected with Labview . To test maximum power density polarization curves were made simultaneously for each section. These were made by disconnecting anode and cathode for 30 minutes to reach open cell voltage (OCV) and subsequently connecting anode and cathode over an external load of 900, 500, 250, 100, 10, 100, 250, 500 and 900 Ω, for 10 minutes each. At day 320 anode was sampled at 9 sample-points, which were evenly distributed over the three anode-sections, so three sample-points in each anode-section. Samples of 1 ml each were measured for conductivity  and pH .-2 s-1, measured at the top of the set-ups witha light intensity meter (Photodyne 44XLA). The climate chamber was controlled at 25°C with 75% humidityand a day-night regime of 14:10 hours was used.The set-ups were placed in a climate chamber (Microclima 1750 Snijders) with an average light intensity of 596 ± 161 μmole mAfter 703 days the set-ups were taken apart and root-weight was measured for the three anode-sections per P-MFC.Internal resistance of the P-MFC can be split in several partial internal resistances as in :2), in which Ran = anodic resistance (Ω.m2), Rcat = cathodic resistance (Ω.m2), RT = transport resistance (Ω.m2).Equation 2 Internal resistance in a P-MFC , in which Ean = measured anode potential at a certain external resistance (V), i = current density (A/m2).Equation 3 Anodic resistance in a P-MFC .Since the theoretical anode potential in the P-MFC is based on a mixed potential  and concCathodic resistance can be calculated from the cathode over-potential according to :2), in which Ecat = measured cathode potential at a certain external resistance (V), i = current density (A/m2).Equation 4 Cathodic resistance in a P-MFC   and can mem = measured potential over the membrane (V), EΔpH = pH gradient loss (V), Eionic = ionic loss (V).Equation 5 Transport loss in a P-MFC (V), in which; EWhen dividing this equation by current, transport resistance can be calculated. The potential over the membrane is measured as the difference between the reference electrode in the anode and the reference electrode in the cathode. The pH resistance is calculated as :cath = cathode pH, pHan = anode pH, and i = current density per membrane area.Equation 6 pH resistance in an MFC, in which pHEquation for pH resistance is derived from the Nernst equation:Equation 7 Nernst-equation, in which This equation shows that one pH-unit difference will lead to a change in anode potential of 59 mV.Ionic resistance is calculated as ,23:an = average distance between point of proton production and membrane, and σan = conductivity of anolyte.Equation 8 Ionic resistance in the anode of an MFC, in which dPower densities are expressed per geometric planting area because it gives insight in the possibility to use this technology for large scale electricity production.P-MFC: Plant-Microbial Fuel Cell the technology to produce electricity with living plants; MFC: Microbial Fuel Cell bio-electrochemical system to produce electricity from organic matter; EMF: Electromotive force driving force for electrons; CEM: Cation exchange membrane selective membrane for cations; OCV: Open Cell Voltage voltage at open circuit (zero current).Two of the authors of the manuscript, Marjolein Helder and David Strik, founded a spin-off company from Wageningen University to further develop the Plant-Microbial Fuel Cell into products. The company does not generate any income and has no interest in the conducted research. Results presented in this paper were not in any way influenced by the existence of the spin-off company.MH co-designed and co-executed the experiment, analyzed data and drafted the paper, DS co-designed and co-executed the experiment, critically assessed data and critically reviewed the paper, HH invented the technology, co-designed the experiment, critically assessed data and critically reviewed the paper, CB designed the research project, critically assessed data and critically reviewed the paper. All authors read and approved the final manuscript.


Anti-TNF therapy has been shown to reduce radiographic joint damage in rheumatoid arthritis (RA) independent of clinical response. This has previously not been examined for periarticular bone loss, the other characteristic feature of bone involvement in RA.The objective of this study was to examine if treatment with the TNF-α inhibitor adalimumab also could reduce periarticular bone loss in RA patients independent of disease activity.RA patients were recruited from the PREMIER study and included 214 patients treated with methotrexate (MTX) plus adalimumab and 188 patients treated with MTX monotherapy. Periarticular bone loss was assessed by digital X-ray radiogrammetry metacarpal cortical index (DXR-MCI). Change in DXR-MCI was evaluated in patients with different levels of clinical response, as assessed by changes in DAS28 score at 52 weeks and in mean C-reactive protein (CRP) levels during follow-up.In the MTX group, there was a greater median DXR-MCI loss among patients with moderate and high disease activity compared to those in remission or with low disease activity . In contrast, periarticular bone loss was independent of disease activity  in the combination group. In the MTX group patients with a mean CRP of ≥ 10 mg/l lost significantly more DXR-MCI than patients with low CRP  whereas in the combination group no significant differences between the two CRP groups was seen .Adalimumab in combination with MTX reduces periarticular bone loss independently of clinical response. These results support the hypothesis that TNF-α stimulates the osteoclast not only by the inflammatory pathway but do also have a direct effect on the osteoclast.NCT001195663ClinicalTrials (NCT):  In rheumatoid arthritis (RA), bone damage on radiographs is visible as erosions and periarticular osteoporosis. Substantial data support that both erosions and osteoporosis in RA share a common cellular pathway which involves stimulation of the osteoclast. This osteoclast activation depends on stimulation from receptor activator of nuclear factor-κ ligand (RANKL) which binds to the receptor activator of nuclear factor-κ (RANK) on the osteoclast. The expression of RANKL is stimulated by pro-inflammatory cytokines , IL-6 and IL-17). In addition recent data also suggest decreased osteoblast activation through the Wnt system [In comparison to disease modifying anti-rheumatic drugs (DMARDs) including methotrexate (MTX), anti-TNF therapy has been shown to be superior in reducing the rate of both radiographic joint damage -4 and haThe objective of this study was to examine if treatment with the TNF-α inhibitor adalimumab also could reduce periarticular bone loss in RA patients independent of disease activity.The PREMIER study cohort was used to examine the relationship between periarticular bone loss and clinical response in RA patients treated with MTX and anti TNF-therapy.In this cohort, radiographic joint progression has recently been reported to be reduced independently of patients' clinical responses to anti-TNF therapy with adalimumab .The clinical, radiographic and bone density data from this 2-year, multi-centre, double-blind, randomised controlled study has previously been described in detail ,9 In shoDigital X-ray radiogrammetry (DXR)  was used to measure hand metacarpal cortical index (MCI) on the same digitised hand X-rays used for assessment of radiographic joint damage. DXR-MCI is defined as the combined cortical thickness divided by the bone width and is a relative bone measure independent of bone size and bone length ,11. In tcombxcxVPA(1-p), where c is a density constant, VPA is volume per area, and p is porosity) was intended to be the main outcome measure in this study. However, many radiographs could not be analysed for BMD because of unknown image resolution. The equation for DXR-BMD is based on volume per area and requires a known resolution. Thus, DXR-MCI, which is a relative measure less dependent of image resolution, was used as the primary outcome measure [DXR-BMD  at 52 weeks. Levels achieved for DAS28 scores were stratified according to the EULAR improvement criteria  for remiDXR-MCI bone loss at 52 weeks follow- up were compared for patients in remission and low disease activity vs. moderate and high disease activity according to treatment groups, as well as for patients in remission, low, moderate and high disease activity.DXR-MCI loss dependent on inflammation, assessed by CRP during follow-up, was also analysed according to treatment groups. Mean CRP was calculated from values at baseline, 26 weeks and 52 weeks follow-up, and a mean CRP of <10 was defined as low inflammation.Since the data were skewed, non-parametric analyses were conducted. No imputations were performed. Baseline values were compared between treatment groups with the Mann-Whitney method for continuous variables and the chi-squared method for categorical variables. DXR-MCI loss is described as negative values. Group analyses were performed with the Mann-Whitney method for two independent samples and Kruskall-Wallis for more than two independent samples. Spearman correlation analyses were conducted in an attempt to correlate changes in DXR-MCI with disease activity measured by DAS28. Analyses that came out with a p-value ≤ 0.05 were considered as statically significant.The PREMIER study was approved a central institutional review board or independent ethics committee at each participating site approved the study, and all patients provided written informed consent .Baseline characteristics are shown in Table In the MTX group there were a significantly greater DXR-MCI loss in the patients with elevated disease activity as assessed by DAS28 than patients in remission and low disease activity, median (interquartile range) (-3.3% (-6.0% to -1.4%) vs. -2.2% (-4.4% to -0.7%), p = 0.01). In the combination group there were no differences in DXR-MCI loss between patients with moderate and high disease activity vs. patient in remission or with low disease activity (-1.9% (-4.7% to -0.7%) vs. -2.4% (-4.1 to -0.5%), p = 0.99), and the levels of loss were closer to those observed for patients in the MTX group in remission . In the group who still had active disease, there was significantly greater median loss in the MTX group  as compared to the combination group.The DXR-MCI loss across subgroups in remission or with low, moderate and high disease activity is shown in Table In the MTX group 184 patients had CRP measures at baseline, 26 and 52 weeks and in the combination group the number was 206. In the MTX group there were 57 patients with mean CRP < 10 mg/L and 127 with CRP ≥10 mg/L. In the combination group the respective values were 86 and 120. The median DXR-MCI loss in the MTX group was significantly higher among patients with high CRP than low CRP (-3.1% (-6.0 to -1.4) vs. -1.9% (-3.9 to -0.2), p <0.01). In the combination group there was no difference regarding median DXR-MCI loss dependent on CRP level (-2.4% (-4.6% to -0.7%) vs. -2.0% (-4.1% to 0.8%), p = 0.48), Figure The main finding in this study was that adalimumab in combination with MTX reduces hand bone loss independently of clinically assessed disease activity. This has previously been shown for radiographic joint damage ,8, but tThese results support the hypothesis that TNF-α can influence bone loss not only by stimulating RANKL by inflammation. The most probable additive mechanism is that TNF-α activates the osteoclast directly by binding to osteoclasts precursors through TNF-α receptor. Blocking of this receptor will down regulate the osteoclast formation ,17. AnotDespite that bone loss was independent of disease activity in the combination group, there were still a bone loss observed. This loss may have been a result of the patient selection in the PREMIER study. The included patients had high disease activity and poor prognosis in terms of bone damage as Rheumatoid Factor-positive and erosive disease ,18.This is a retrospective study and we did not have any influence on the technical conditions regarding the radiographs. Due to difficulties analysing DXR-BMD the relative measure DXR-MCI was used, as described in detail previously . HoweverWe conclude that adalimumab in combination with MTX reduces hand bone loss independent of disease activity. This study highlights the bone protecting effect of anti-TNF-α therapy.BMD: bone mineral density; CRP: C-reactive protein; DAS28: disease activity score based on 28 joint count; DMARDs: disease modifying anti-rheumatic drugs; DXR: digital X-ray radiogrammetry; ESR: erythrocyte sedimentation rate; HAQ: health assessment questionnaire; IL: interleukin; MCI: metacarpal cortical index; MTX: methotrexate; RA: rheumatoid arthritis; RANK: receptor activator of nuclear factor-κ; RANKL: receptor activator of nuclear factor-κ ligand; TNFα: tumour necrosis factor alphaM. Hoff, T. K. Kvien, A. Kavanaugh and G. Haugeberg have received consulting fees as speakers from Abbott Laboratories. Tore K. Kvien, A. Kavanaugh and G. Haugeberg have received founding for independent research from Abbott Laboratories. Aake Elden is employed by Abbott Laboratories. Johan Kälvesten is employed by Sectra.MH performed statistical analyses and prepared the manuscript. TKK contributed to statistical analysis and interpretation of results. JK analysed the radiographs. AE prepared the collection of radiographs. AK contributed to statistical analysis and interpretation of results. GH contributed to statistical analysis and interpretation of results and was the main responsible investigator of this project. All the authors substantially contributed to the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/12/54/prepub